RESEARCH ARTICLE 1863 Development 139, 1863-1873 (2012) doi:10.1242/dev.074005 © 2012. Published by The Company of Biologists Ltd Identification of molecular compartments and genetic circuitry in the developing mammalian kidney Jing Yu1,2, M. Todd Valerius1,*, Mary Duah1, Karl Staser1, Jennifer K. Hansard1, Jin-jin Guo1, Jill McMahon1, Joe Vaughan1, Diane Faria1, Kylie Georgas3, Bree Rumballe3, Qun Ren2, A. Michaela Krautzberger1, Jan P. Junker4, Rathi D. Thiagarajan3, Philip Machanick3,‡, Paul A. Gray5,§, Alexander van Oudenaarden4, David H. Rowitch6, Charles D. Stiles7, Qiufu Ma5, Sean M. Grimmond3, Timothy L. Bailey3, Melissa H. Little3 and Andrew P. McMahon1,¶ SUMMARY Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations. KEY WORDS: Genome-scale expression screen, Transcriptional regulator, Kidney, Mouse INTRODUCTION that cap each branch tip (Carroll et al., 2005; Kobayashi et al., The mammalian metanephric kidney is essential for the 2008; Park et al., 2007). At each round of branching, a subset of maintenance of water and electrolyte homeostasis, the regulation these progenitors undergoes an epithelial transition that establishes of blood pressure and blood cell composition, and bone formation a renal vesicle (RV). RVs undergo elaborate patterning and (Koeppen and Stanton, 2001). Development of a functional kidney morphogenesis along a glomerular-collecting duct axis that is requires a complex interplay among its principal cellular crucial for organ function. A highly developed vascular network components (Costantini and Kopan, 2010; Dressler, 2009). (Gomez et al., 1997; Woolf and Loughna, 1998), interstitial support The nephric duct-derived ureteric epithelium forms the arborized cells and a variety of distinct subregions with specialized properties network of the collecting duct system in response to branching that include contractile and sensory functions all contribute to the signals derived from adjacent mesenchyme. This network is crucial physiological actions of the kidney. for urine transport from the nephron to the ureter, and the Not surprisingly, given the central role of the kidney in human maintenance of pH and osmolarity in tissue fluids. Signals from the health, the experimental analysis of mammalian kidney nascent ureteric epithelium regulate survival, proliferation and development has received considerable attention. The kidney was differentiation of mesenchymal, stem cell-like, nephron progenitors the first mammalian organ shown to replicate a developmental program in culture (Grobstein, 1953; Grobstein, 1955; Saxen, 1987). Since those pioneering studies in the 1950s, a wealth of 1Department of Stem Cell and Regenerative Biology, Department of Molecular and genetic, molecular and cellular information has informed on the Cellular Biology, Harvard Stem Cell Institute, Harvard University, 16 Divinity Avenue, control of kidney development (Costantini and Kopan, 2010; Cambridge, MA 02138, USA. 2Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. 3Institute for Molecular Dressler, 2009). Collectively, these studies have underscored the Bioscience, University of Queensland, St Lucia 4072, Australia. 4Department of complexity of kidney development, and highlighted the need for a Physics and Biology, Massachusetts Institute of Technology, 77 Massachusetts more systematic analysis of cell types, cell relationships and cell 5 Avenue, Cambridge, MA 02139, USA. Department of Neurobiology, Harvard interactions in the assembly of the kidney. The increasing evidence Medical School, Boston, MA 02115, USA. 6Departments of Pediatrics and Neurological Surgery, Howard Hughes Medical Institute, University of California San that developmental deficiencies increase the risk of kidney disease Francisco, 35 Medical Center Way, San Francisco, CA 94143, USA. 7Department of in later life emphasizes the need to enhance our understanding of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, developmental events (Bertram et al., 2011; Song and Yosypiv, USA. 2011). *Present address: Department of Surgery, Transplant Institute, Beth Israel Deaconess One barrier to progress is a dearth of molecular markers that can Medical Center, Harvard Medical School, Boston, MA 02115 USA distinguish cell types and facilitate analysis of developmental ‡Present address: Department of Computer Science, Rhodes University, Grahamstown, 6140, South Africa programs. In many systems, transcriptional regulators have served §Present address: Department of Anatomy and Neurobiology, Washington University as important cell type-specific markers and drivers of cell School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110-1093, USA specification and differentiation. For example, large-scale ¶Author for correspondence ([email protected]) expression screens of transcriptional regulators have proven fruitful Accepted 1 March 2012 in identifying organizational and developmental features in the DEVELOPMENT 1864 RESEARCH ARTICLE Development 139 (10) assembly of the mammalian nervous system (Gray et al., 2004). In situ hybridization of whole-mount urogenital systems with We surmised that systematic identification of the expression digoxigenin-labeled riboprobes patterns of a large set of transcriptional regulators in the developing For whole-mount in situ hybridization, E15.5 urogenital system samples urogenital system would provide new insights into the emergence were rehydrated through a graded series of methanol/NaCl. After of cell heterogeneity central to kidney formation and function. proteinase K (10 mg/ml) treatment for 30 minutes, samples were fixed Furthermore, the accrued data could potentially facilitate in silico again and prehybridized at 70°C for 1 hour before being hybridized with 500 ng/ml digoxigenin-labeled riboprobes at 70°C overnight. All steps prediction of gene networks, and enable the design of novel genetic post-hybridization were performed with a BioLane HTI liquid handling approaches for analysis of key cell types. system (Intavis Bioanalytical Instruments AG, Widdersdorferstrasse, Here, we report on a genome-scale in situ analysis of the Koeln, Germany). Briefly, samples were washed with hot solutions at 65°C expression of genes encoding mammalian transcriptional and treated with 100 mg/ml RNase A for 1 hour. After additional stringent regulatory factors in the developing mouse urogenital system. Our hot washes, samples were washed with 1ϫMBST [0.1 M maleic acid, 0.15 analysis provides a wealth of new markers of kidney development M NaCl, 0.1% Tween-20 (pH 7.5)], blocked in blocking solution (10% and reveals novel molecular subdomains in developing renal sheep serum in MBST plus 2% Boehringer Mannheim Blocking Reagent) structures. A meta-analysis of selected transcriptional regulators for 3-4 hours at room temperature, and incubated subsequently with anti- highlights the potential of this dataset for discovery of networks digoxigenin antibody conjugated with alkaline phosphatase (AP) (Roche, governing differentiation of terminal cell fates. Indianapolis, IN, USA, 1:4000) overnight at 4°C. Extensive washing in 1ϫMBST was followed by incubation in NTMT [100 mM NaCl, 100 mM MATERIALS AND METHODS Tris-HCl (pH 9.5), 50 mM MgCl2, 0.1% Tween-20, 2 mM levamisole). Further details of the protocols described below can be found Samples were then removed from the BioLane HTI, and the hybridization at http://www.gudmap.org/Research/Protocols/McMahon.html and signal visualized by adding BM purple. Color reactions were terminated at http://www.gudmap.org/Research/Protocols/Little.html. All experimental 3-hour, 6-hour, 9-hour, 12-hour, 24-hour, 36-hour and 48-hour time points, procedures with mice were performed in accordance with institutional and when signals were strong or a background developed. After development national animal welfare laws, guidelines and policies, and were approved of the colorimetric assay, samples were post-fixed, cleared through a by relevant institutional animal care committees. graded series of glycerol and stored in 80% glycerol/PBS. Images were then captured with a Nikon DXM1200 digital camera (Nikon, Melville, Plasmid preparation and riboprobe production NY, USA), attached to a Nikon SMZ1500 stereoscope (Nikon, Melville, Plasmid glycerol stocks from the Brain Molecular Anatomy Project NY, USA) at two different magnifications to view the entire urogenital (BMAP) cDNA